It is well understood that, in the use of display and selection technologies, access to greater diversity allows for a more effective selection of molecules with the highest affinity, specificity, stability, and/or other desirable characteristics.
Past methods that have been developed include phage display, ribosome display, CIS display, and mRNA display. Recently, there has been increased interest in identifying molecules using in vivo screening (J Control Release. 2003 Aug. 28; 91(1-2):183-6). This approach has been made possible using phage display, but suffers from the limited diversity afforded by the phage display technologies. Ribosome or mRNA display would fail for this application due to the instability of the RNA species. Therefore, development of DNA-protein fusions would be highly desirable, due to the increased stability of the species.
Three types of DNA-protein fusions have been described. CIS display is one method where coupled in vitro transcription/translation is used to generate a dsDNA binding protein that covalently binds to the DNA as it is being transcribed and translated (PNAS, 101(9): 2806-2810). However, one of the primary limitations of this technology is that the synthesized protein can bind to any neighboring DNAs that are nearby during the transcription/translation process, resulting in mis-tagged fusions. The second method is that of Kurz and Lohse (Chembiochem. 2001 Sep. 3; 2(9):666-72). This method involves formation of covalent adducts with mRNA using a multifunctional species that can covalently bond with translated protein, create a ribosomal pause at the covalent adduct on RNA, and serve as a primer for reverse transcription. A limitation of this method is the inefficiency of the covalent linking step with RNA using psoralen. The third method is that of Yonezawa et al. (Nucleic Acids Res. 2003 Oct. 1; 31(19): e118.) In this method, DNA encoding streptavidin and a region of diverse peptides is biotinylated, placed in a synthetic microsphere with translation machinery and translated such that the streptavidin (tetrameric) will bind to the biotinylated DNA. The limitation of this method is that the resultant species is tetrameric, which can be troublesome for affinity selections due to multiple binding species on one particle (rebinding effect).
Herein is described a simple, efficient method for generating nucleic acid protein fusions is required which may employ noncovalent attachment between the nucleic acid and the protein, and which will allow the formation of DNA-protein fusions.